Mechanisms of protection against alpha-synuclein-induced oxidative stress

α-突触核蛋白诱导的氧化应激的保护机制

基本信息

批准号：
7759196
负责人：
Stephan N. Witt
金额：
$ 27.91万
依托单位：
LOUISIANA STATE UNIV HSC SHREVEPORT
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-03-01 至 2013-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7759196
关键词：
14-3-3 Proteins Abbreviations Alanine Amino Acids Apoptosis Binding Biochemical Brain Cell Cycle Cell Death Cells Centromere Chemicals Code Complement Cytosol Dopamine Dyes Enzymes Equilibrium Exhibits Functional disorder Galactose Genes Glycogen (Starch) Synthase Goals Green Fluorescent Proteins Homologous Gene Human Ion Channel Knock-out Lead Letters Libraries Membrane Mitochondria Modification Monitor Mutation Neurons Nomarski Interference Contrast Microscopy Oxidative Stress Parkinson Disease Pharmaceutical Preparations Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Production Protein Binding Proteins Reactive Oxygen Species Regulator Genes Role Saccharomyces cerevisiae Signal Transduction Signaling Molecule Signaling Protein Source Staining method Stains Sucrose System Testing Therapeutic Toxic effect Vesicle Yeasts alpha synuclein base cDNA Library cytochrome c design dihydrorhodamine 123 early onset in vivo mitochondrial dysfunction multicatalytic endopeptidase complex mutant novel peroxisome phosphoneuroprotein 14 prevent research study respiratory trafficking yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand the mechanism by which phosphorylation of alpha-synuclein (1-syn) enhances the toxicity of this neuroprotein. Employing a yeast system, we seek to extend our findings that a highly conserved phosphatase and two highly conserved signaling proteins prevent 1-syn-induced ROS and cell death. The specific aims are to: 1) Determine whether ROS originate from mitochondria or peroxisomes by expressing 1-syn in respiratory-deficient cells or peroxisome-deficient cells and staining with an ROS-sensitive dye. 2) Determine the mechanism by which phosphorylation of 1-syn at S129 enhances 1-syn's ability to cause cell death. A two-hybrid screen will be employed to find proteins that interact with the toxic phosphorylated form of WT 1-syn. 3) Characterize how cells die when the kinase/phosphatase balance shifts in favor of kinases. Although the essential phosphatase that we discovered that protects cells from 1-syn-induced ROS cannot be deleted, its non-essential regulatory subunits can be. Experiments will use GFP-tagged 1-syns to determine whether 1-syn inclusion formation, proteasome dysfunction, or mitochondrial dysfunction causes cell death when the kinase/phosphatase balance is tipped in favor of the kinases by knocking out phosphatase regulatory genes. 4) We discovered two yeast signaling genes that exhibit synthetic lethal interactions with WT 1-syn and A30P but not A53T. These signaling genes are also present in human neurons where they regulate the cell cycle, intracellular signaling, differentiation, ion-channels, vesicle trafficking, and apoptosis. We hypothesize that these two signaling proteins bind to toxic phosphorylated forms of 1- syn (WT or A30P), and this protects cells from the build up of 1-syn. This hypothesis will be tested by monitoring for direct binding between the various 1-syns and the signaling protein and by monitoring how cells die when the signaling genes are deleted. Understanding how kinases/phosphatases and signaling molecules regulate 1-syn phosphorylation state and hence its toxicity could lead to novel neuroprotective therapeutics that could delay or even prevent the onset of PD. Such therapeutics would modulate the kinase-phosphatase equilibrium in such a way as to drive 1-syn into a dephosphorylated state.7. Project Narrative A simple chemical modification (phosphorylation) of the Parkinson's disease-related protein alpha- synuclein dramatically increases the toxicity of alpha-synuclein. Our goal is to determine the enzymes and proteins that regulate the chemical modification of alpha-synuclein. This information could lead to novel drugs that function to inhibit the chemical modification of alpha-synuclein.

描述（由申请人提供）：该项目的长期目的是了解α-突触核蛋白（1-Syn）磷酸化增强该神经蛋白质毒性的机制。我们采用酵母系统，试图扩展发现高度保守的磷酸酶和两种高度保守的信号蛋白可以防止1-Syn诱导的ROS和细胞死亡。具体目的是：1）确定ROS是否来自线粒体或过氧化物酶体是通过在呼吸缺陷的细胞中表达1-Syn或过氧化物酶体缺陷的细胞，并用ROS敏感的染料染色。 2）确定S129处1-Syn磷酸化的机制增强了1-Syn导致细胞死亡的能力。将使用两杂交筛选来找到与WT 1-Syn的毒性磷酸化形式相互作用的蛋白质。 3）表征当激酶/磷酸酶平衡转移而有利于激酶时，细胞如何死亡。尽管我们发现保护细胞免受1-SYN诱导的ROS的必需磷酸酶不能被删除，但其非必需的调节亚基可以是。实验将使用GFP标记的1-SYNS来确定当激酶/磷酸酶平衡倾向于通过敲除磷酸蛋白酶调节基因而倾向于激酶时，当激酶/磷酸酶平衡倾向于激酶时，是否会导致细胞死亡。 4）我们发现了两个酵母信号基因，它们与WT 1-Syn和A30p表现出合成的致命相互作用，但不是A53T。这些信号基因也存在于人类神经元中，它们调节细胞周期，细胞内信号传导，分化，离子通道，囊泡运输和凋亡。我们假设这两种信号蛋白与1- SYN（WT或A30P）的有毒磷酸化形式结合，这可以保护细胞免受1-Syn的堆积。该假设将通过监视各种1-Syns和信号蛋白之间的直接结合以及监测信号基因删除时细胞的死亡方式来检验。了解激酶/磷酸酶和信号分子如何调节1-Syn磷酸化状态，因此其毒性可能导致新型的神经保护疗法，从而延迟甚至可以阻止PD发作。这种治疗剂将以将1-Syn驱动到去磷酸化状态的方式来调节激酶 - 磷酸酶平衡7。项目叙述帕金森氏病α-突触核蛋白的简单化学修饰（磷酸化）显着增加了α-突触核蛋白的毒性。我们的目标是确定调节α-核蛋白化学修饰的酶和蛋白质。这些信息可能导致新型药物的作用，以抑制α-突触核蛋白的化学修饰。